phosphorylation of the tomato Β-subunit of the snrk complex by adi3 and its possible role in sugar...
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Phosphorylation of the TomatoΒ-Subunit of the SnRK Complex by Adi3 and its
Possible Role in Sugar Signaling
JULIAN AVILA-PACHECO
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Protein Phosphorylation
One of the most ubiquitous types of post-translational modifications
Ser, Thr, Tyr
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Protein Phosphorylation
STRUCTURE
ACTIVITY
LOCALIZATION
PROTEIN STABILITY
INTERACTION PARTNERS
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Adi3
(i.e. VIGS)
Adi3
Adi3 is a kinase involved in cell deathADI3 VIRAL INDUCED
GENE SILENCING (VIGS)
Dev
aren
ne e
t al.
(200
6) E
MB
O J
.
How does Adi3Regulate cell death?
Silencing the expression of Adi3 causes spontaneous cell death in tomato
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(β) (γ)
(α)
SNF1 Related Protein Kinase Complex
SNF (Sucrose Non Fermenting)
AMPK (AMP Dependent-Kinase)
AKIN/SNRKAdi3
Adi3 interacts with the tomato SnRK complex
The SnRK complex is highly conserved across eukaryotes
MASTER REGULATOR OF ENERGY HOMEOSTASIS
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(β) (γ)
(α)?
METABOLICHOMEOSTASIS
STRESSSIGNALING
?
The SnRK complex (Mammals vs. Plants)
Adi3 Cell Death Regulation
GERMINATIONIN SRUCROSE
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(β) (γ)
(α)
Adi3
Adi3 phosphorylates β at Ser26
P32
(β)
Adi3
(β)
Adi3
Ser26Ala
Replacing serine 26 with alanine blocks phosphorylation by Adi3.
?
Ser26Ala
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(β)
Experimental design
Karlheinz Knoch
(β)Ala
- Generate two transgenic plants over-expressing β and βAla using the plant model organism Arabidopsis thaliana using Agrobacterium mediated transformation.- Assess cell death phenotypes- Estimate germination efficiency under high, normal, or low concentration of sucrose.
GFP GFP
Does phosphorylation of β- play a role in SnRK function in vivo?
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(β)
Transgenic plants express β-GFP
Karlheinz Knoch
(β)Ala
GFP GFPA. thaliana
WT β βAla
< β-GPF
α-GFP
The expression of β-subunit in transgenic plants was tested using western blot with α-GFP Antibodies.
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Transgenic plant cell death phenotype
wt
β
βAla - Line 1
βAla – Line 2
No significant differences in plant development or viability were observed.
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Transgenic germination on sucrose
0mM 29mM 200mM
WT, β, βAla
wt
β
βAla1
βAla2
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Transgenic germination on high sucrose
200mM
wt
β
βAla1
βAla2
High sucrose inhibits cotyledon expansion
β over-expression causes aberrant phenotypes
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Conclusions
- We successfully produced Arabidopsis plants over-expressing wild type and phosphorylation deficient β.
- No cell death related phenotype was observed in phosphorylation deficient β Plants.
- Plants over-expressing β phosphorylation deficient mutants appear unaffected by germination inhibition in sucrose.
(β) (γ)
(α)
Adi3
SnRK complex regulation.